Development and Characterization of Potent Cyclic Acyldepsipeptide Analogues with Increased Antimicrobial Activity

Abstract

The problem of antibiotic resistance has prompted the search for new antibiotics with novel mechanisms of action. Analogues of the A54556 cyclic acyldepsipeptides (ADEPs) represent an attractive class of antimicrobial agents that act through dysregulation of caseinolytic protease (ClpP). Previous studies have shown that ADEPs are active against Gram-positive bacteria (e.g., MRSA, VRE, PRSP (penicillin-resistant Streptococcus pneumoniae)); however, there are currently few studies examining Gram-negative bacteria. In this study, the synthesis and biological evaluation of 14 novel ADEPs against a variety of pathogenic Gram-negative and Gram-positive organisms is outlined. Optimization of the macrocyclic core residues and N-acyl side chain culminated in the development of 26, which shows potent activity against the Gram-negative species Neisseria meningitidis and Neisseria gonorrheae and improved activity against the Gram-positive organisms Staphylococcus aureus and Enterococcus faecalis in comparison with known analogues. In addition, the co-crystal structure of an ADEP-ClpP complex derived from N. meningitidis was solved.

Imipridone Anticancer Compounds Ectopically Activate the ClpP Protease and Represent a New Scaffold for Antibiotic Development

“…The Gram-positive species E. faecium was sensitive to ONC212 (MIC = 62.5-125 mM), whereas most Gram-negative species were insensitive toward ONC212 or ONC201. A notable exception was N. gonorrhoeae, which was sensitive to ONC212 (MIC = 31.25-62.5 mM), similar to the previously observed sensitivity of Neisseria species toward ADEP analogs (Goodreid et al 2016). The mycobacteria M. smegmatis (MIC = 125-250 mM) and the attenuated M. tuberculosis H37Ra strain (MIC = 250-500 mM) were mildly sensitive to ONC201 but not ONC212, perhaps due to the more complex ClpP1/ClpP2 subunit structures in these species (Table 2).…”

“…For example, imipridone-based anti-cancer therapy might adversely impact the microbiome or, conversely, the use of imipridones as antibiotics might negatively impact normal tissues that require high mitochondrial capacity. A number of compounds have been identified as ClpP activators, including the ADEPs and a variety of (semi)-synthetic analogs (Brötz-Oesterhelt et al 2005;Carney et al 2014b;Goodreid et al 2016;Wong et al 2018;Griffith et al 2019), ADEP-based fragments (Carney et al 2014a), the imipridones and the related TR series compounds (Allen et al 2013;Graves et al 2019;Ishizawa et al 2019;Wong and Houry 2019), the natural product sclerotiamide (Lavey et al 2016), and the synthetic compounds D9 (Stahl et al 2018) and ACP1 (Leung et al 2011). X-ray structures show that ONC201, ADEPs, and D9 all occupy the same binding site in human CLPP (Stahl et al 2018;Wong et al 2018;Ishizawa et al 2019;Wong and Houry 2019).…”

Bacterial proteases, untapped antimicrobial drug targets

“…39,62 Numerous other synthetic chemistry efforts have explored motifs to overcome these limitations, but most have resulted in diminished activity (Figure 4c). 54,[63][64][65][66][67] Given the intimate binding of ADEP in its hydrophobic pocket, this result is perhaps predictable and suggests that modification on the ADEP scaffold may have reached an impasse.…”

“…51 Insight into this loss of regulation has been provided by crystal structures of ADEP-activated ClpP from E. coli, B. subtilis, M. tuberculosis and Neisseria meningitidis (Figure 4b). [52][53][54][55] An ADEP molecule binds to each monomer in the ClpP tetradecamer in the same hydrophobic pocket that is used by the AAA+ ATPases, thus inhibiting their interaction (Figure 4d). 52,55,56 Binding mimics the ATPase's conformational control of ClpP, inducing alignment of the serine catalytic triad and a rigid body rotation of the ClpP monomers, widening the axial pore from 10-12 Å to 20 Å in E. coli.…”

“…51,54 Semisynthetic ADEP derivatives are Bacterial proteases, untapped antimicrobial drug targets E Culp and GD Wright effective against Enterococcus faecalis, S. aureus and S. pneumoniae in mouse and rat models with activity superior to linezolid, 51 and activity against MRSA in a murine periotinitis model is superior to vancomycin. 58 Notably, resistance to ADEPs develop at a frequency as high as 10 − 6 in these bacterial species by mutations diminishing the function of ClpP, which is non-essential.…”